The ectodermal dysplasias (EDs) are congenital, diffuse, and nonprogressive. The EDs comprise a large, heterogeneous group of inherited disorders that share primary defects in the development of 2 or more tissues derived from embryonic ectoderm. To date, more than 192 distinct disorders have been described. The most common EDs are X-linked hypohidrotic ectodermal dysplasia (Christ-Siemens-Touraine syndrome) and hidrotic ectodermal dysplasia (Clouston syndrome). The X-linked hypohidrotic ectodermal dysplasia is also known and hereinafter referred to as XLHED.
The tissues primarily involved are the skin, hair, nails, eccrine glands, and teeth. A reduction in the number of hair follicles in conjunction with structural hair shaft abnormalities may be seen.
Structural hair shaft abnormalities may result from aberrations in hair bulb formation and include longitudinal grooving, hair shaft torsion, and cuticle ruffling. Hair bulbs may be distorted, bifid, and small.
Eccrine sweat glands may be absent or sparse and rudimentary, particularly in patients with hypohidrotic ED.
Hypoplasia of the salivary and lacrimal glands may occur. In some patients, mucous glands may be absent in the upper respiratory tract and in the bronchi, esophagus, and duodenum.
Abnormal morphogenesis or absence of teeth may occur.
Abnormal nail plate formation may result in brittle, thin, ridged, or grossly deformed nails.
The mortality rate in early infancy (below 2 years old) approaches 30%. Morbidity and mortality is related to the absence or dysfunction of eccrine and mucous glands. Beyond early childhood, life expectancy ranges from normal to slightly reduced.
An activator protein, the ectodysplasin isoform A1 (Eda-A1, hereinafter referred to as EDA1) which is required for normal development of several ectodermally derived organs in humans and mice has been identified. This molecule is coded by the ectodysplasin gene (EDA) and belongs to the tumor necrosis factor family. The ectodysplasin gene codes for 2 proteins isoforms, EDA1 and EDA2, which bind to and activate two different receptors, EDA1 receptor (EDAR) and X-linked EDA1 receptor (XEDAR), respectively. The EDA1 receptor is hereinafter referred to as EDAR.
XLHED is characterized by the absence or the functional deficiency of EDA1, the ligand of EDAR.
Studies have been performed to characterize the origin of XLHED and to find appropriate treatments. Most of these studies have been done on Tabby mice which share many symptoms with human XLHED patients. The phenotype of Tabby mice, like in human XLHED patients, is caused by mutations in the EDA gene located on the X chromosome.
Different approaches have been studied to treat XLHED.
One of these approaches is the use of recombinant proteins containing the receptor-binding domain of Eda-A1 fused to the C-terminus of an IgG1 Fc domain. The publication “Permanent correction of an inherited ectodermal dysplasia with recombinant EDA”, O. Gaide et al., Nature Medicine, vol. 9, number 5, pp 614-618, May 2003, describes the administration of recombinant EDA1 to developing embryos and newborn Tabby mice in order to correct the phenotype and provide a basis for a possible treatment of XLHED.
Such an approach is also described in US Patent 2005/152,872 (Gaide et al.). In particular this document discloses a recombinant fusion protein containing an amino-acid sequence which comprises: (a) the Fc section or part of an Fc section of an immunoglobulin as component (A) or a functional variant of component (A); (b) the extracellular part of a TNF ligand or a partial sequence of the extracellular part of a TNF ligand as component (B) or a functional variant of component (B); and optionally (c) a transition area between component (A) and component (B), containing a linker.
Another approach is disclosed in U.S. Pat. No. 6,355,782 (Zonana et al.) and U.S. Pat. No. 7,115,555 (Zonana et al.). In these documents a nucleic acid sequence encoding a human EDA1, methods and compositions for increasing or decreasing the development of cells or tissues of ectodermal origin, such as hair, teeth, skin, and/or sweat glands, by altering EDA1 activity in a cell or tissue are described. EDA1 activity can be increased or decreased using the EDA1, dl and DL gene, cDNA and protein sequences (and variants, polymorphisms and mutants thereof), as well as anti-sense molecules and specific binding agents disclosed herein alone or with a pharmaceutical carrier.
Considering that antibodies are the most widely used type of therapeutic proteins, that the safety, long half-life, good bio-availability, ease of production and controlled cost of manufacturing for the antibodies are apparently well established, such an approach was envisioned in US 2003/0023991 (Zonana et al.). In particular this document describes DNA and amino acid sequences for the protein ligand (EDA1-II) and receptors (dl and DL) involved in ectodermal dysplasia. Also disclosed are variant DNA and amino acid sequences, and therapeutic applications of the ligands and receptors. Also described are different potential applications in which administration of antibody against EDAR (dl/DL) may be used. This application in particular describes the use of antagonists against dl/DL (EDAR receptor). According to this application, these antagonists can be used to reduce hair growth, for example in the treatment of hirsutism, to inhibit tooth development, such as ectopic teeth, to selectively eliminate sweat glands, for example on the upper lip or under the arm, and to inhibit breast epithelial cell proliferation, for example in the treatment of breast cancer or other trauma of the skin. Finally, the production and use of monoclonal or polyclonal antibodies are envisioned. However, no specific sequences of antibodies against EDAR either agonists or antagonists are described in this document. No working examples are provided in order to evidence that monoclonal or polyclonal antibodies are biologically active, effective and functional.
It might be stated that the selection of monoclonal antibodies is a routine work for a person skilled in the art. However, one cannot acknowledge that the preparation and obtainment of isolated agonist anti-EDAR monoclonal antibodies was routine and does not involve inventive step. For example, the CH11 monoclonal antibody directed against human Fas, another TNF receptor family member, was obtained by immunization of mice with membranes of FS-7 human fibroblasts (Yonehara et al, 1989, J. Exp. Med 169. 1747-1756). CH11 is an IgM. When an IgG1 recognizing the exact same epitope (mAb ZB4) or a divalent Fab′2 of CH11 was used, there was no agonist activity (Fadeel et al, 1997, Int Immunol 9, 201-209). A second example of an agonist monoclonal antibody against human Fas is APO-1. The APO-1 monoclonal antibody against human Fas was obtained by immunizing mice with plasma membrane of the human SKW 6.4 cell line (Trauth et al, 1989, Science 245, 301-305). This antibody is an IgG3. Upon isotype switch, this antibody looses its agonist activity (Dhein et al, 1992, J. Immunol. 149, 3166-3173), although the Fas-binding portion of the antibody remains the same. This result was tentatively explained by the propensity of IgG3 to self-aggregate. These two examples indicate that the obtainment of agonist monoclonal antibodies against a TNF receptor family member is absolutely not trivial to a skilled in the art.
In the context of the present invention, Applicants had to face several problems as follows:
                Commercial polyclonal anti-EDAR antibodies (raised in the goat, from R&D Systems) failed to induce death of EDAR:Fas-transduced Jurkat cells. One could believe that the polyclonal antibody preparations were made of a mixture of antibodies with various activities (agonist, antagonist, etc.). It was therefore Applicant's disappointment to observe that the cell-based assay did not reveal any agonist activity, even when using a large quantity of antibody. These results were not encouraging for the development of agonist anti-EDAR monoclonal antibodies.        The development of an EDA1-dependent biological assay was found difficult for several reasons. First, there are only few cell lines, which endogenously express EDAR (one example is the human keratinocyte cell line HaCat). Several human, mouse and rat keratinocyte cell lines were screened for EDAR expression. This was done by staining cells with Fc-EDA1 and the anti-EDAR mAb, as detected by flow cytometry. None of the cell lines was found to express detectable levels of EDAR. Second, an N{tilde over (F)} B-dependent read-out system was found not satisfying (phosphorylation and degradation of I B), because it was neither quantitative, nor fully reproducible. In order to overcome these limitations, it was finally attempted to introduce in EDAR-transfected fibroblasts a reporter gene (e.g. luciferase) under the control of the N{tilde over (F)} B promoter. However, this system was also found not to result in a biological activity assay working properly (low signal to noise ratio and low sensitivity). Therefore the use of the techniques known to the skilled in the art was not found satisfactory.        The naturally occurring soluble EDA1 protein is probably a multimer of trimers (Swee et al, J. Biol. Chem. 284:27267-76, 2009), and Fc-EDA1 is an hexameric ligand. As it has been extensively documented for Fas, the Applicants have obtained evidence that the simultaneous engagement and clustering of multiple EDAR receptors was necessary for biological activity (Swee et al, J. Biol. Chem. 284:27267-76, 2009). By contrast, anti-EDAR antibodies are only divalent. It was therefore not likely that this molecular format would be as biologically active as Fc-EDA1.        No convenient animal model was available. Without the identification of a convenient strain of mice (EDAR-deficient mice), the development of antibodies according to the invention would have been considerably hampered. It is assumed that in the absence of Applicant's proposed animal model and specific preparation process, anti-EDAR monoclonal antibodies with agonist properties would have not been obtained (i.e. because they would have had distinct binding sites on EDAR).        
However, there is a need for more effective treatment for XLHED and for increasing the quality of life of those suffering from this disease. In particular no curative treatment of children, young adults or adults suffering from ectodermal dysplasia such as XLHED or tooth agenesis is presently available.
The aim of the present invention is to provide a new process for the preparation of substantially purified and isolated agonist anti-EDAR monoclonal antibodies that are biologically active, effective and functional. The present invention also allows to rapidly discriminating the best agonist anti-EDAR monoclonal antibodies. The present invention also demonstrates that the use of agonist anti-EDAR monoclonal antibodies represent drug candidates for the treatment of the XLHED or related diseases.
These and other objects as will be apparent from the foregoing have been achieved by the present invention.